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Experiments

• Open Loop [Aa]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL

ii) K12Z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=50 ng/mL and inoculum=1,0.1 uL/mL

iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB

iv) The contents of the flask with OD=0.217 were filtered out.

v) 7 dilutions were made (full range of IPTG conc) for pL.LuxR.Y.pR.C with AI from 50 ng/mL aTc and new 2xM9 in 1:1 ratio.

• Open Loop [Cc]

i) Cells were transferred to 50 mL Glu M9 in 250 mL flask with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL

ii) K12z1 was inoculated in 5 mL Glu M9 in 50 mL tube with [aTc]=5 ng/mL and inoculum=1,0.1 uL/mL

iii) pL.LuxR.Y.pR.C, K12z1, pL.Y were inoculated in LB.

iv) The contents of the flask with OD=0.103 were filtered out.

v) 7 dilutions over the full range of IPTG were made for pL.LuxR.Y.pR.C with AI from 5 ng/mL aTc and new 2xM9 in 1:1 ratio

• Control experiments:
  • -ve control: K12z1
  • Inoculated positive control:
    • pL.YFP; 1xGlu M9; 500 uM IPTG
    • I726081; K12Z1 in aTc (50 ng/mL) medium and new 2xGlu M9 in 1:1 ratio; 500 uM IPTG
    • I726081; only new 1xGlu M9; 500 uM IPTG

Microscopy

• The open loop samples [Aa] were imaged and FACS was done.

• The open loop samples [Cc] were imaged and FACS was done.

• The open loop samples [Dd] were imaged and FACS was done.

Analysis

Today we compiled the data collected from all our open-loop experiments performed at various concentrations of aTc (ng/ml). The following matrix of information was obtained:

CFP mean values		IPTG (uM) or luxR.YFP
		0	5	10	50	100	500	1000
aTc (ng/ml) or luxI.CFP	0
	1
	5
	10
	20
	50

YFP mean values		IPTG (uM) or luxR.YFP
		0	5	10	50	100	500	1000
aTc (ng/ml) or luxI.CFP	0
	1
	5
	10
	20
	50

These values, obtained from the microscopy experiments, give us a clear idea of the equivalence between luxI.CFP and luxI.YFP. A scatter plot with each dot representing a cell was also plotted to see the variation of fluorescence with the change in aTc and IPTG concentrations. Red : 0 ng/ml aTc Green : 1 ng/ml aTc Blue : 5 ng/ml aTc Cyan : 10 ng/ml aTc Magenta : 20 ng/ml aTc Yellow : 50 ng/ml aTc

CFP Fluorescence	YFP Fluorescence
CFP fluorescence	YFP fluorescence
CFP fluorescence	YFP fluorescence
Surface plots : The increasing values along the luxI.CFP axis are the increasing concentrations of aTc whereas the increasing values along the luxR.YFP axis are the increasing concentrations of IPTG.